Previous experiments have shown that substitutions of some amino acids in a conserved region of the vaccinia virus DNA polymerase are allowed. Vaccinia virus containing the altered polymerase formed plaques similar to those of wild-type virus. To ultimately identify what aspect of DNA polymerase function was associated with the structure of this particular conserved region, it is desirable to overexpress the enzyme. If modest amounts of functional enzyme can be expressed from a cloned gene, then the in vivo substitutions can be tested in vitro. Attempts to express substantial amounts of even a portion of the enzyme in bacteria were not successful. However, others have shown that a functional vaccinia virus DNA polymerase can be overexpressed in tissue culture cells infected with a recombinant vaccinia virus. To follow that lead and improve on that type of expression system, I am attempting to insert the polymerase gene into a new vaccinia virus vector that does not require an additional virus or plasmid to achieve overexpression. To accomplish this, I first inserted the entire gene into the DNA of the replicative form of M13mp19 bacteriophage. The resulting phage contains a recombinant DNA with one strand of the polymerase gene. This construct allows the application of an improved form of the Eckstein mutagenesis procedure in which the in vitro mutagenic manipulations can be completed in one day. After several manipulations to remove internal NdeI restriction enzyme target sites, a unique NdeI site was installed at the locus of the first codon of the polymerase. This construct was inserted into the vaccinia virus transfer vector pVote2, and the recombinant DNA was isolated. This DNA is designed to transfer the polymerase gene by homologous recombination into the hemagglutinin region of the vaccinia virus vector. If the recombination is successful and the resulting virus causes infected cells to express ten times more functional polymerase than is produced with wild-type virus infection, then the supply of altered polymerase should be sufficient to conveniently conduct in vitro enzyme assays.